Apical macropinocytosis in polarized MDCK cells: Regulation by N-ethylmaleimide-sensitive proteins

In cells tested so far endocytosis seems to be dependent on N-ethylmaleimid e (NEM)-sensitive proteins, and treatment with NEM results in a complete bl ock of endocytosis. We here demonstrate that treatment of polarized MDCK I cells with NEM strongly increased endocytosis of ricin and horseradish pero xidase at the apical side, and electron microscopy revealed NEM-induced for mation of large macropinosomes at the apical pole. The NEM-stimulated apica l endocytosis seemed to involve phosphatidylinositol-3 kinase, protein kina se C and phospholipase D and it was dependent on ATP. Moreover, in contrast to endocytosis in nonpolarized cells ricin endocytosis at the basolateral side continued in the presence of NEM whereas endocytosis of transferrin wa s blocked. Furthermore, recycling of ricin endocytosed in the absence of NE M was not inhibited on either side upon addition of NEM demonstrating the e xistence of a NEM-resistant fusion machinery. The results suggest that the fusogenic property of both the apical and the basolateral plasma membrane o f MDCK cells differs from that typically observed in cells unable to polari ze.